The complete nucleotide sequence of the sixth gene encoding VP6 of two subgroup I animal rotavirus (vervet monkey rotavirus SA-11 and bovine rotavirus RF) and one subgroup II human rotavirus (Wa) was determined previously. Comparison of the deduced amino acid sequences indicated a high degree of homology; more than 90% of the amino acids were conserved between subgroup I and subgroup II rotaviruses. In the present study we deduced the amino acid sequence of the VP6 protein of 4 additional rotavirus stains, each with a different subgroup specificity: human rotavirus strain 1076 (subgroup I), porcine rotavirus strain Gottfied (subgroup (II), equine rotavirus stained H-2 (neither subgroup I nor II) and equine rotavirus strain FI-14 (both subgroup I and II). This allowed us to compare the VP6 amino acid sequences of rotaviruses with dual or no known subgroup reactivity with those of rotaviruses belonging to subgroup I or II. This approach was taken in an attempt to identify epitopes responsible for subgroup specificity. In addition, the importance of the trimeric conformation of the VP6 protein to subgroup epitopes was investigated. Most of the VP6 genes the different strains contain a unique AUG. In contrast, the VP6 gene of H-2 strain contains an additional AUG at residue 132-134, which results in the translation of a second VP6 protein. Both VP6 products assume a trimeric conformation in the mature virion.